Glutamine synthetase (GS) is intimately involved in brain physiology, recycling from glial cells as glutamine to neurons the glutamate released at the synapses. Glutamate, glutamine, and Gamma-amino-butyric acid (GABA) are among the most abundant free amino acids in brain, Glu and GABA having excitory and inhibitory functions, respectively. Mn(II), known to be essential in trace amounts for normal CNS function, has been found to be tightly bound to the octameric brain GS, as Mn4E [Wedler, Denman, and Roby (1982) Biochemistry 24, 6389]. Whether Mn(II) is bound to GS in vivo is not yet proven, since crude cell extracts contain a "Mn(II)-exchange activator protein," (MEAP) whereas pure Mn4E in vitro fails to exchange bound Mn(II) with radioactive 54Mn(II). It is proposed to study the regulation of brain GS by Mn(II) and other ligands by: (1) Purification of the MEAP and studying its interaction with Mn4E in terms of how it alters kon, koff, and Kd for bound Mn(II), as well as the possibility that it is the main regulation vector for brain GS, responding to signals such as cyclic-AMP. (2) In-depth biophysical studies of the environments of bound Mn(II) at the K1, K2, and K3 sites: E = Mn4E = Mn4EMn4 = Mn4EMn4Mn8 K1 K2 K3 using pulsed NMR (PRR), 17-0 superhyperfine ESR, and laser-induced lanthamide luminescence: namely determining the number of exchangeable waters, the distance between the K1 and K2 sites, the exact coordination mode of ATP to each Mn(II), as well as the stoichiometry and binding constants for Mg(II) are the goals of this work. (3) The nature of the induced cooperativity effects of AMP on ATP binding will be probed further by steady-state kinetics. (4) In vivo studies will be undertaken to prove or disprove the existence and regulatory importance of Mn4E. The methods to be used are direct EM metal-specific probes or automicroradiography (EM) to locate Mn(II) ions, whereas immunocytochemical methods are envisioned for GS localization. This latter area is exploratory in nature.